Abstract
Plants consist of many functionally specialized cell types, each with its own unique epigenome, transcriptome, and proteome. Characterization of these cell type-specific properties is essential to understanding cell fate specification and the responses of individual cell types to the environment. In this chapter we describe an approach to map chromatin features in specific cell types of Arabidopsis thaliana using nuclei purification from individual cell types with the INTACT method (isolation of nuclei tagged in specific cell types) followed by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). The INTACT system employs two transgenes to generate affinity-labeled nuclei in the cell type of interest, and these tagged nuclei can then be selectively purified from tissue homogenates. The primary transgene encodes the nuclear tagging fusion protein (NTF), which consists of a nuclear envelope-targeting domain, the green fluorescent protein, and a biotin ligase recognition peptide, while the second transgene encodes the E. coli biotin ligase (BirA), which selectively biotinylates NTF. Expression of NTF and BirA in a specific cell type thus yields nuclei that are coated with biotin and can be purified by virtue of their affinity for streptavidin-coated magnetic beads. Compared with the original INTACT nuclei purification protocol, the procedure presented here is greatly simplified and shortened. After nuclei purification, we provide detailed instructions for chromatin isolation, shearing, and immunoprecipitation. Finally, we present a low input ChIP-seq library preparation protocol based on the nano-ChIP-seq method of Adli and Bernstein, and we describe multiplex Illumina sequencing of these libraries to produce high quality, cell type-specific epigenome profiles at a relatively low cost. The procedures given here are optimized for Arabidopsis but should be easily adaptable to other plant species.
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References
Bailey-Serres J (2013) Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers. Annu Rev Plant Biol 64:293–325
Wang D, Mills ES, Deal RB (2012) Technologies for systems-level analysis of specific cell types in plants. Plant Sci 197:21–29
Rogers ED, Jackson T, Moussaieff A, Aharoni A, Benfey PN (2012) Cell type-specific transcriptional profiling: implications for metabolite profiling. Plant J 70:5–17
Galbraith DW, Birnbaum K (2006) Global studies of cell type-specific gene expression in plants. Annu Rev Plant Biol 57:451–475
Deal RB, Henikoff S (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev Cell 18:1030–1040
Deal RB, Henikoff S (2011) The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6:56–68
Palovaara J, Saiga S, Weijers D (2013) Transcriptomics approaches in the early Arabidopsis embryo. Trends Plant Sci 18:514–521
Steiner FA, Talbert PB, Kasinathan S, Deal RB, Henikoff S (2012) Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res 22:766–777
Amin NM, Greco TM, Kuchenbrod LM, Rigney MM, Chung MI, Wallingford JB, Cristea IM, Conlon FL (2014) Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT). Development 141:962–973
Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, Garcha J, Winte S, Masson H, Inagaki S, Federici F, Sinha NR, Deal RB, Bailey-Serres J, Brady SM (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol. doi: dx.doi.org/10.1104/pp.114.239392
Adli M, Bernstein BE (2011) Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq. Nat Protoc 6:1656–1668
Adli M, Zhu J, Bernstein BE (2010) Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat Methods 7:615–618
Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192
Acknowledgements
This work was supported by funds from Emory University. We thank Marko Bajic, Kristopher Hite, Paja Sijacic, and Shannon Torres for helpful suggestions on improving the manuscript.
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Wang, D., Deal, R.B. (2015). Epigenome Profiling of Specific Plant Cell Types Using a Streamlined INTACT Protocol and ChIP-seq. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_1
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DOI: https://doi.org/10.1007/978-1-4939-2444-8_1
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